Neuropeptides influence mood, sensation, learning and memory, and the function of peripheral organs. Their release typically begins slowly only after bursts of electrical activity and is limited so that distal terminals are not easily emptied. Our goal is to understand how these unique properties are generated. Initially, we used live cell imaging of calcium and a GFP-tagged neuropeptide/hormone along with patch clamping to study peptide release by cultured cells. These experiments demonstrated great diversity in the handling of peptidergic dense core vesicles (DCVs). We then generated transgenic animals to study DCV dynamics in synaptic boutons. In vivo experiments with Drosophila synapses suggest that calcium influx, triggered by depolarization or electrical activity, increases DCV motion within boutons to facilitate recruitment for neuropeptide release. Furthermore, we generated a new fluorescent protein construct to measure the delay in release following fusion pore formation, and have produced transgenic flies that express this protein. Also, we generated animals for inducible expression of fluorescent DCVs. Finally, we began to study the basis of regulated DCV motion. We are now poised to study processes that could govern the time and activity dependence of neuropeptide release. Aim 1 will determine the activity and calcium dependence of DCV motion within synaptic boutons and neuropeptide release. Aim 2 examines the mechanistic basis for regulated DCV movement within boutons. Aim 3 will determine whether the releasable DCVs in synapses are replenished first by refractory DCVs that were already present in boutons, or by new DCVs transported into the bouton from the axon. Aim 4 will detect the initial fusion with the plasma membrane to learn about the location and speed of neuropeptide exocytosis. Understanding DCV dynamics is essential for determining how neuropeptide release is uniquely controlled. This could provide a basis for controlling secretion of neuropeptides that are involved in pain, mood, hunger, and sleep